U.S. patent number 7,875,613 [Application Number 11/554,056] was granted by the patent office on 2011-01-25 for tetracyclic inhibitors of cysteine proteases, the pharmaceutical compositions thereof and their therapeutic applications.
This patent grant is currently assigned to Hybrigenics SA. Invention is credited to Frederic Colland, Matteo Colombo, Laurent Daviet, Etienne Formstecher, Philippe Guedat, Xavier Jacq, Jean-Christophe Rain.
United States Patent |
7,875,613 |
Guedat , et al. |
January 25, 2011 |
Tetracyclic inhibitors of cysteine proteases, the pharmaceutical
compositions thereof and their therapeutic applications
Abstract
The present invention concerns new compounds of formula (I),
their process of preparation and their therapeutic use ##STR00001##
wherein R3, R4, R5, R6, Y, Het1, T, U, V, W, X, Ru, Rv and Rw are
as defined in claim 1.
Inventors: |
Guedat; Philippe (Montenois,
FR), Jacq; Xavier (Etuz, FR), Colland;
Frederic (Puiseux En France, FR), Daviet; Laurent
(Antony, FR), Formstecher; Etienne (Paris,
FR), Rain; Jean-Christophe (Ermont, FR),
Colombo; Matteo (Camnago, IT) |
Assignee: |
Hybrigenics SA (Paris,
FR)
|
Family
ID: |
39331049 |
Appl.
No.: |
11/554,056 |
Filed: |
October 30, 2006 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20080103149 A1 |
May 1, 2008 |
|
Current U.S.
Class: |
514/243; 544/184;
435/184 |
Current CPC
Class: |
A61P
29/00 (20180101); A61P 35/00 (20180101); A61P
31/00 (20180101); C07D 487/04 (20130101); A61P
25/16 (20180101); A61P 25/28 (20180101) |
Current International
Class: |
A61K
31/53 (20060101); C07D 487/04 (20060101); C12N
9/99 (20060101) |
Field of
Search: |
;514/243 ;544/184
;435/184 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Aulakh; Charanjit S
Attorney, Agent or Firm: Schulman; B. Aaron Wright; Terry L.
Stites & Harbison PLLC
Claims
The invention claimed is:
1. A compound having a formula: ##STR00046## wherein : is either a
single or double bond, as appropriate ; is either none or a single
bond, as appropriate; T, U, W, X are the same or different and are
chosen from C or N; V is N; Y is N-OR1, NR'1, CR2R'2; R1 is H,
Alkyl, Alkenyl, Alkoxyalkyl, Aryloxyalkyl, Arylalkyl,
Alkoxycarbonylalkyl, Carboxyalkyl; R'1 is H, Alkyl, Aryl or
Aralkyl; R2, R'2 are each the same or different and are
independently selected from H, Alkyl, Aryl or Aralkyl; Rv is
absent; Rw is either H or absent; Ru is absent or is chosen from
the group consisting of H, CN, .dbd.O, Hal, Alk, OAlk, OH, NRCN,
C(CN).dbd.C(OH)(OAlk), SR, NRR', C(O)NRR', Heterocycle, Aryl,
Heteroaryl, Cycloalkyl, where Alk, Aryl, Heteroaryl, Heterocycle,
Cycloalkyl are optionally substituted by Hal, NRR', CN, OH,
CF.sub.3, Aryl, Heteroaryl , OAlk; R3, R4, R5, R6 are each
identical or different and are independently chosen from the group
consisting of H, OAlk, Alk, Hal, NRR', CN, OH, OCF.sub.3, CF.sub.3,
Aryl, Heteroaryl; R and R' are each identical or different and are
independently chosen from the group consisting of H, Alk, wherein
Alk is optionally substituted by Hal, NRR', CN, OH, CF.sub.3, Aryl,
Heteroaryl; or their pharmaceutically acceptable salts, or their
optical isomers, racemates, diastereomers or enantiomers, or their
regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
2. The compound according to claim 1, wherein Ru is chosen from H,
Aryl, Alk, NRR', Hal, -AlkAryl, -AlkOH, -AlkOAlk, Cycloalkyl.
3. The compound according to claim 1, wherein R3, R4, R5, R6 are
each identical or different and are independently chosen from the
group consisting of H, Hal, Alk, OAlk, OCF.sub.3.
4. The compound according to claim 1, wherein they are of formula
(Ia): ##STR00047## wherein R3, R4, R5, R6, Y, T, U, V, W, X, Ru are
as defined in anyone of the preceding claims.
5. The compound according to claim 1, chosen from the group
consisting of: 3-Methyl-1,2,3a,4,
10-pentaaza-cyclopenta[b]fluoren-9-one O-methyl-oxime
3-Methyl-1,2,3a,4, 10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Methyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 3-Butyl-1,2,3a,4,
10-pentaaza-cyclopenta[b]fluoren-9-one O-allyl-oxime
1-Butyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-decyl-oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
[1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-ylidene
]-phenyl-amine
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
lithium salt, or their pharmaceutically acceptable salts, or their
optical isomers, racemates, diastereomers or enantiomers, or their
regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
6. A process of preparation of a compound according to claim 1,
comprising the step of reacting a corresponding compound of formula
(I'): ##STR00048## wherein R3, R4, R5, R6, T, U, V, W, X, Ru, Rv,
Rw are defined as in claim 1, and wherein each of Ru', Rv', Rw' is
similar to Ru, Rv, Rw or is a precursor group of corresponding Ru,
Rv, Rw, and wherein the step of reacting a corresponding compound
of formula (I') comprises one or more steps allowing a precursor
group to be transformed into the desired Ru, Rv or Rw group, and
optionally isolating the compound according to claim 1.
7. A process of preparation of a compound according to claim 1,
comprising the step of reacting corresponding compounds of formula
(II) and (III): ##STR00049## wherein R3, R4, R5, R6, T, U, V, W, X,
Ru, Rv, Rw are defined as in claim 1.
8. The process according to claim 7, wherein the reaction is
carried out in an organic protic solvent in the presence of an
acid.
9. A pharmaceutical composition comprising a compound having a
formula ##STR00050## wherein R3, R4, R5, R6, T, U, V, W, X, Ru, Rv
and Rw are as defined in claim 1.
10. A method for inhibiting one or more cysteine proteases,
comprising administering a compound as defined in claim 1 to a
patient in the need thereof, wherein the one or more cysteine
proteases are selected from the group consisting of USP5, USP7,
USP8, UCH-L3, and Cathepsine B.
Description
The present invention concerns new inhibitors of cysteine
proteases, their process of preparation and their therapeutic
use.
Proteases can be categorized based on their substrate specificities
or mechanisms of catalysis. Upon the basis of the mechanism of
peptide hydrolysis, five major protease classes are known: serine,
cysteine, aspartic, threonine and metallo-proteases. Cysteine
proteases comprise, inter allia, de-ubiquitination enzymes,
caspases, cathepsins, calpains as well as viral, bacterial or
parasitic cysteine proteases.
De-ubiquitination enzymes include Ubiquitin Specific Proteases
(USPs) and Ubiquitin Carboxy Hydrolases (UCHs). Broadly speaking,
the ubiquitin pathway regulates protein degradation and is more
particularly involved in cancer, in neurodegenerative diseases such
as Alzheimer's disease, Parkinson's disease, in inflammation, in
viral infectivity and latency (in particular for Herpes simplex
virus-1, Epstein-Barr virus, SARS coronavirus), or in
cardiovascular diseases (Chem. Rev. 1997, 97, p. 133-171; Chem.
Rev. 2002, 102, p. 4459-4488; J. Biochem. 2003, 134, p. 9-18; J.
Virology, 2005, 79(7), p. 4550-4551; Cardiovasc. Res. 2004, 61, p.
11-21).
Caspases have been shown to be involved in apoptosis and hence are
targets in hepatitis, liver failure, inflammation, cardiac ischemia
and failure, renal failure, neurodegeneration, deafness, diabetes,
or stroke (J. Pharmacol Exp. Ther., 2004, 308(3), p. 1191-1196, J.
Cell. Physiol., 2004, 200(2), p. 177-200; Kidney Int, 2004, 66(2),
p. 500-506; Am. J. Pathol., 2004, 165(2), p. 353-355; Mini Rev.
Chem., 2004, 4(2), p. 153-165; Otol. Neurotol., 2004, 25(4), p.
627-632; Ref. 7, 21, 22, 23, 24, 25).
Cathepsins generally have been shown to be involved in cancer and
metastasis, inflammation, immunology/immunoregulation (Eur. Respir.
J., 2004, 23(4), p. 620-628) and atherosclerosis (Ageing Res. Rev.
2003, 2(4), p. 407-418). More particularly, cathepsins include
cathepsin B and B-like which are implicated in cancer and
metastasis, and arthritis (Cancer Metastasis Rev., 2003, 22(2-3),
p. 271-286; Biol. Chem., 2003, 384(6), p. 845-854 and Biochem. Soc.
Symp., 2003, 70, p. 263-276), cathepsin D, involved in particular
in cancer and metastasis (Clin. Exp. Metastasis, 2004, 21(2), p.
91-106), cathepsin K acting in osteoporosis and arthritis (Int. J.
Pharm., 2004, 277(1-2), p. 73-79), cathepsin S which has been shown
to play a role in antigen presentation in immunology (Drug News
Perspective, 2004,17(6), p. 357-363).
Calpains play a role in ageing in general (Ageing Res. Rev. 2003,
2(4), p. 407-418), as well as diabetes (Mol. Cell. Biochem., 2004,
261(1), p. 161-167) and cataract (Trends Mol. Med., 2004,10(2), p.
78-84) more particularly.
Viral cysteine proteases have been identified in rhinoviruses,
poliomyelitis virus, hepatitis A virus, hepatitis C virus,
adenovirus, or SARS coronavirus (Chem. Rev. 1997, 97, p. 133-171;
Chem. Rev. 2002, 102, p. 4459-4488; J. Virology, 2005, 79(7), p.
4550-4551 and Acta Microbiol. Immunol. Hung., 2003, 50(1), p.
95-101).
Bacterial cysteine proteases include streptopain, staphylococcal
cysteine protease, clostripain or gingipains; yeasts such as
Aspergillus flavus have also been shown to express cysteine
proteases which may constitute a virulence factor (Chem. Rev. 1997,
97, p. 133-171).
Parasitic cysteine proteases have been reviewed in Molecular &
Biochemical Parasitology (2002, 120, p. 1-21) and Chem. Rev. (2002,
102, p. 4459-4488) for example. It is worth noting that the
parasitic agents responsible for most major parasitic diseases are
making use of their own cysteine proteases at some point or another
of their infective, nutritive or reproductive cycles; such diseases
include malaria, Chagas' disease, African trypanosomiasis,
leishmaniasis, giardiasis, trichomoniasis, amoebiasis,
crypto-sporidiasis, toxoplamiasis, schistosomiasis, fasciolasis,
onchocercosis, and other infections by some other flat or round
worms.
Therefore, identifying a novel class of inhibitors of cysteine
proteases is of significant importance in a wide range of diseases
and pathological conditions.
U.S. Pat. No. 6,514,927, WO01/79209 and WO02/02562 disclose
compounds comprising 4 fused cycles. However, their use as cysteine
protease inhibitors is not suggested.
According to a first object, the present invention concerns a
compound of formula (I):
##STR00002## wherein:
is either a single or double bond, as appropriate;
is either none or a single bond, as appropriate;
##STR00003## is a 5 to 7-membered heterocycle, preferably
heteroaryl comprising 1 to 5 heteroatoms optionally substituted by
one or more substituents chosen from the group consisting in H, CN,
.dbd.O, Hal, Alk, OAlk, OH, NRCN, C(CN).dbd.C(OH)(OAlk), SR, NRR',
C(O)NRR', Heterocycle, Aryl, Heteroaryl, where Alk, Aryl,
Heteroaryl, heterocycle are optionally substituted by Hal, NRR',
CN, OH, CF.sub.3, Aryl, Heteroaryl, OAlk; where
##STR00004## are fused together by T and X; Y is N--OR1, NR'1,
CR2R'2; R1 is H, Alkyl, Alkenyl, Alkoxyalkyl, Aryloxyalkyl,
Arylalkyl, Alkoxycarbonylalkyl, Carboxyalkyl; R'1 is H, Alkyl, Aryl
or Aralkyl; R2, R'2 are each the same or different and are
independently selected from H, Alkyl, Aryl or Aralkyl; T, U, V, W,
X are the same or different and may be chosen from C, N, O, S. Ru,
Rv, Rw are the same or different and may be chosen from the group
consisting in H, CN, .dbd.O, Hal, Alk, OAlk, OH, NRCN,
C(CN).dbd.C(OH)(OAlk), SR, NRR', C(O)NRR', Heterocycle, Aryl,
Heteroaryl, Cycloalkyl where Alk, Aryl, Heteroaryl, heterocycle,
Cycloalkyl are optionally substituted by Hal, NRR', CN, OH,
CF.sub.3, Aryl, Heteroaryl , OAlk. R3, R4, R5, R6 are each
identical or different and are independently chosen from the group
consisting in H, OAlk, Alk, Hal, NRR', CN, OH, OCF.sub.3, CF.sub.3,
Aryl, Heteroaryl; R and R' are each identical or different and are
independently chosen from the group consisting in H, Alk, wherein
Alk is optionally substituted by Hal, NRR', CN, OH, CF.sub.3, Aryl,
Heteroaryl; or their pharmaceutically acceptable salts, hydrates,
or hydrated salts, or the polymorphic crystalline structures of
these compounds or their optical isomers, racemates, diastereomers
or enantiomers, or their regioisomers, geometrical isomers (E and
Z) or mixtures thereof.
Preferably, T, U, V, W, X are C or N.
Preferably, Y is N--OR1 or NR'1, more preferably N--OR1, notably
N--OH, N-Alkyl, N--OAlkenyl, N--OAlkyl-O-Alkyl,
N--O-Alkyl-CO--OAlkyl, N--O-Alkyl-COOH.
It will be appreciated that when Y is CR2R2', R2 and/or R2' cannot
form a fused ring with the rest of the structure of formula
(I).
Preferably,
##STR00005## contains 2 or 3 heteroatoms; more preferably, 2 or 3
N.
Most preferably,
##STR00006## is unsubstituted.
Preferably, Ru, Rv, Rw is chosen from H, Aryl, Alk, NRR', Hal,
-AlkAryl, -AlkOH, -AlkOAlk, Cycloalkyl.
Preferably,
##STR00007## where Rw is H.
Preferably, R3, R4, R5, R6 are each identical or different and are
independently chosen from the group consisting in H, Hal, Alk,
OAlk, OCF.sub.3.
Preferably, R and R' are each identical or different and are
independently chosen from the group consisting in H, Alk.
Preferably, Rv, Rw are either H or absent.
Preferred compounds of formula (I) are those of formula (Ia):
##STR00008##
Most preferred compounds are notably those of formulae (I.sub.1) to
(I.sub.4)
##STR00009##
Preferred compounds of the invention are chosen from the group
consisting in:
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Methyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Butyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-decyl-oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
[1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-ylidene]-phenyl-ami-
ne
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
lithium salt,
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts, or the polymorphic crystalline structures of these compounds
or their optical isomers, racemates, diastereomers or enantiomers,
or their regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
Most preferred compounds are notably selected from the group
consisting in:
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-decyl-oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
lithium salt,
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts, or the polymorphic crystalline structures of these compounds
or their optical isomers, racemates, diastereomers or enantiomers,
or their regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
As used hereabove or hereafter:
Alk represents alkyl, alken or alkyn.
"Alkyl" means an aliphatic hydrocarbon group which may be straight
or branched having 1 to 20 carbon atoms in the chain. Preferred
alkyl groups have 1 to 12 carbon atoms in the chain. "Branched"
means that one or more lower alkyl groups such as methyl, ethyl or
propyl are attached to a linear alkyl chain. Exemplary alkyl groups
include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,
n-pentyl, 3-pentyl, octyl, nonyl, decyl.
"Alken" means an aliphatic hydrocarbon group containing a
carbon-carbon double bond and which may be straight or branched
having 2 to 15 carbon atoms in the chain. Preferred alkenyl groups
have 2 to 12 carbon atoms in the chain; and more preferably about 2
to 4 carbon atoms in the chain. Exemplary alkenyl groups include
ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl,
n-pentenyl, heptenyl, octenyl, nonenyl, decenyl.
"Alkyn" means an aliphatic hydrocarbon group containing a
carbon-carbon triple bond and which may be straight or branched
having 2 to 15 carbon atoms in the chain. Preferred alkynyl groups
have 2 to 12 carbon atoms in the chain; and more preferably 2 to 4
carbon atoms in the chain. Exemplary alkynyl groups include
ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl,
n-pentynyl, heptynyl, octynyl and decynyl.
"Alkoxyalkyl" means an alkyl-O-alkyl group wherein the alkyl groups
are independently as defined herein. An example of alkoxyalkyl is
methoxyethyl.
"Alkoxycarbonylalkyl" means an alkyl-O--CO-alkyl-group wherein the
alkyl groups are independently as defined herein. Exemplary alkoxy
carbonyl alkyl groups include methoxy- and ethoxy-carbonyl methyl
and carbonyl ethyl groups.
"Halogen atom" refers to fluorine, chlorine, bromine or iodine
atom; preferably fluorine and chlorine atom.
"Aryl" means an aromatic monocyclic or multicyclic hydrocarbon ring
system of 6 to 14 carbon atoms, preferably of 6 to 10 carbon atoms.
Exemplary aryl groups include phenyl or naphthyl.
"Arylalkyl" means an aryl-alkyl-group wherein the aryl and alkyl
groups are as defined herein. An example of arylalkyl groups is
benzyl.
"Aryloxyalkyl" mean an aryl-O-alkyl-group wherein the alkyl and
aryl groups are as defined herein. An exemplary aryloxyalkyl group
is phenoxypropryl.
As used herein, the terms "heterocycle" or "heterocyclic" refer to
a saturated, partially unsaturated or unsaturated, non aromatic
stable 3 to 14, preferably 5 to 10 membered mono, bi or multicyclic
rings wherein at least one member of the ring is a hetero atom.
Typically, heteroatoms include, but are not limited to, oxygen,
nitrogen, sulfur, selenium, and phosphorus atoms. Preferable
heteroatoms are oxygen, nitrogen and sulfur.
Suitable heterocycles are also disclosed in The Handbook of
Chemistry and Physics, 76.sup.th Edition, CRC Press, Inc.,
1995-1996, p. 2-25 to 2-26, the disclosure of which is hereby
incorporated by reference.
Preferred non aromatic heterocyclic include, but are not limited to
pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl,
tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl, dioxanyl,
dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl,
imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl,
tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydro-pyranyl,
tetrahydropyranyl, dihydropyranyl, tetrahydro-pyridyl,
dihydropyridyl, tetrahydropyrinidinyl, dihydrothiopyranyl,
azepanyl, as well as the fused systems resulting from the
condensation with a phenyl group.
As used herein, the term "heteroaryl" or aromatic heterocycles
refers to a 5 to 14, preferably 5 to 10 membered aromatic hetero,
mono-, bi- or multicyclic ring. Examples include pyrrolyl, pyridyl,
pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl,
quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl,
benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl,
isothiazolyl, triazoyl, tetrazolyl, isoquinolyl, benzothienyl,
isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl,
pyridyl-N-oxide, as well as the fused systems resulting from the
condensation with a phenyl group.
"Carboxyalkyl" means a HOOC-alkyl-group wherein the alkyl group is
as defined herein. Preferred groups include carboxymethyl and
carboxyethyl.
"Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl",
"heterocycle" and the likes refers also to the corresponding
"alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene",
"heteroarylene", "heterocyclene" and the likes which are formed by
the removal of two hydrogen atoms.
As used herein, the term "patient" refers to either an animal, such
as a valuable animal for breeding, company or preservation
purposes, or preferably a human or a human child, which is
afflicted with, or has the potential to be afflicted with one or
more diseases and conditions described herein.
As used herein, a "therapeutically effective amount" refers to an
amount of a compound of the present invention which is effective in
preventing, reducing, eliminating, treating or controlling the
symptoms of the herein-described diseases and conditions. The term
"controlling" is intended to refer to all processes wherein there
may be a slowing, interrupting, arresting, or stopping of the
progression of the diseases and conditions described herein, but
does not necessarily indicate a total elimination of all disease
and condition symptoms, and is intended to include prophylactic
treatment.
As used herein, the term "pharmaceutically acceptable" refers to
those compounds, materials, excipients, compositions or dosage
forms which are, within the scope of sound medical judgment,
suitable for contact with the tissues of human beings and animals
without excessive toxicity, irritation, allergic response or other
problem complications commensurate with a reasonable benefit/risk
ratio.
As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. The
pharmaceutically acceptable salts include the conventional
non-toxic salts or the quaternary ammonium salts of the parent
compound formed, for example, from non-toxic inorganic or organic
acids. For example, such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like; and the salts
prepared from organic acids such as acetic, propionic, succinic,
tartaric, citric, methanesulfonic, benzenesulfonic, glucoronic,
glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric,
maleic, lactic and the like. Further addition salts include
ammonium salts such as tromethamine, meglumine, epolamine, etc.,
metal salts such as sodium, potassium, calcium, zinc or
magnesium.
The pharmaceutically acceptable salts of the present invention can
be synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two. Generally, non-aqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17.sup.th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418,
the disclosure of which is hereby incorporated by reference.
The compounds of the general formula (I) having geometrical
isomers, regioisomers and stereoisomers are also a part of the
invention.
According to a further object, the present invention is also
concerned with the process of preparation of the compounds of
formula (I).
The compounds and process of the present invention may be prepared
in a number of ways well-known to those skilled in the art. The
compounds can be synthesized, for example, by application or
adaptation of the methods described below, or variations thereon as
appreciated by the skilled artisan. The appropriate modifications
and substitutions will be readily apparent and well known or
readily obtainable from the scientific literature to those skilled
in the art.
In particular, such methods can be found in R. C. Larock,
Comprehensive Organic Transformations, Wiley-VCH Publishers,
1999.
It will be appreciated that the compounds of the present invention
may contain one or more asymmetrically substituted carbon atoms,
and may be isolated in optically active or racemic forms. Thus, all
chiral, diastereomeric, racemic forms, isomeric forms of a
structure are intended, unless the specific stereochemistry or
isomeric form is specifically indicated. It is well-known in the
art how to prepare and isolate such optically active forms. For
example, mixtures of stereoisomers may be separated by standard
techniques including, but not limited to, resolution of racemic
forms, normal, reverse-phase, and chiral chromatography,
preferential salt formation, recrystallization, and the like, or by
chiral synthesis either from chiral starting materials or by
deliberate synthesis of target chiral centers.
Additionally, the process of the invention may lead to several
regioisomers which are all encompassed by the present invention.
Regioisomers are generally isolated by chromatography.
Compounds of the present invention may be prepared by a variety of
synthetic routes. The reagents and starting materials are
commercially available, or readily synthesized by well-known
techniques by one of ordinary skill in the arts. All substituents,
unless otherwise indicated, are as previously defined.
In the reactions described hereinafter, it may be necessary to
protect reactive functional groups, for example hydroxy, amino,
imino, thio or carboxy groups, where these are desired in the final
product, to avoid their unwanted participation in the reactions.
Conventional protecting groups may be used in accordance with
standard practice, for examples see T. W. Greene and P. G. M. Wuts
in Protective Groups in Organic Chemistry, 3.sup.rd ed., John Wiley
and Sons, 1999; J. F. W. McOmie in Protective Groups in Organic
Chemistry, Plenum Press, 1973.
Some reactions may be carried out in the presence of a base. There
is no particular restriction on the nature of the base to be used
in this reaction, and any base conventionally used in reactions of
this type may equally be used here, provided that it has no adverse
effect on other parts of the molecule. Examples of suitable bases
include: sodium hydroxide, potassium carbonate, triethylamine,
alkali metal hydrides, such as sodium hydride and potassium
hydride; alkyllithium compounds, such as methyllithium and
butyllithium; and alkali metal alkoxides, such as sodium methoxide
and sodium ethoxide.
Usually, reactions are carried out in a suitable solvent. A variety
of solvents may be used, provided that it has no adverse effect on
the reaction or on the reagents involved. Examples of suitable
solvents include: hydrocarbons, which may be aromatic, aliphatic or
cycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene,
toluene and xylene; amides, such as dimethylformamide; alcohols
such as ethanol and methanol and ethers, such as diethyl ether and
tetrahydrofuran.
The reactions can take place over a wide range of temperatures. In
general, it is found convenient to carry out the reaction at a
temperature of from 0.degree. C. to 150.degree. C. (more preferably
from about room temperature to 100.degree. C.). The time required
for the reaction may also vary widely, depending on many factors,
notably the reaction temperature and the nature of the reagents.
However, provided that the reaction is effected under the preferred
conditions outlined above, a period of from 3 hours to 20 hours
will usually suffice.
The compound thus prepared may be recovered from the reaction
mixture by conventional means. For example, the compounds may be
recovered by distilling off the solvent from the reaction mixture
or, if necessary, after distilling off the solvent from the
reaction mixture, pouring the residue into water followed by
extraction with a water-immiscible organic solvent and distilling
off the solvent from the extract. Additionally, the product can, if
desired, be further purified by various well-known techniques, such
as recrystallization, reprecipitation or the various chromatography
techniques, notably column chromatography or preparative thin layer
chromatography.
The process of preparation of a compound of formula (I) of the
invention is a further object of the present invention.
According to a first aspect, compounds of the invention of formula
(I) can be obtained from reacting corresponding compounds of
formula (II) and (III):
##STR00010## wherein R3, R4, R5, R6, T, U, V, W, X, Ru, Rv, Rw are
defined as in formula (I).
Generally, the reaction is carried out in an organic protic
solvent, such as an alcohol (preferably ethanol), in the presence
of an acid such as acetic acid.
Alternatively and/or cumulatively, compounds of formula (I) may be
obtained from corresponding compounds of formula (I'):
##STR00011##
wherein R3, R4, R5, R6, Het1, T, U, V, W, X, Ru, Rv, Rw are defined
as in formula (I),
wherein each of Ru', Rv', Rw' is similar to Ru, Rv, Rw or is a
precursor group of corresponding Ru, Rv, Rw, by one or more step
allowing a precursor group to be transformed into the desired Ru,
Rv or Rw group.
According to the present invention, the expression "precursor
group" of a functional group refers to any group which can, by one
or more reactions, lead to the desired function, by means of one or
more suitable reagents. Those reactions include de-protection, as
well as usual addition, substitution or functionalization
reactions.
Compounds of formula (I') may be obtained from corresponding
compounds of formula (II) and (III) as discussed above.
Compounds of formula (I) may notably be obtained from compounds of
formula (I') disclosed in EP 05292612.8.
The above reactions can be carried out by the skilled person by
applying or adapting the methods illustrated in the examples
hereinafter.
Further, the process of the invention may also comprise the
additional step of isolating the compound of formula (I). This can
be done by the skilled person by any of the known conventional
means, such as the recovery methods described above.
The starting products (II) and (III) are commercially available or
may be obtained by applying or adapting any known methods or those
described in the examples.
The synthesis may also be carried out in one pot as a
multicomponent reaction.
According to a further object, the present invention concerns also
the pharmaceutical compositions comprising a compound of formula
(I) as defined below:
##STR00012## wherein
is either a single or double bond, as appropriate;
is either none or a single bond, as appropriate;
##STR00013## is a 5 to 7-membered heterocycle, preferably
heteroaryl comprising 1 to 5 heteroatoms optionally substituted by
one or more substituents chosen from the group consisting in H, CN,
.dbd.O, Hal, Alk, OAlk, OH, NRCN, C(CN).dbd.C(OH)(OAlk), SR, NRR',
C(O)NRR', Heterocycle, Aryl, Heteroaryl, where Alk, Aryl,
Heteroaryl, heterocycle are optionally substituted by Hal, NRR',
CN, OH, CF.sub.3, Aryl, Heteroaryl , OAlk; where
##STR00014## are fused together by T and X; Y is N--OR1, NR'1,
CR2R'2; R1 is H, Alkyl, Alkenyl, Alkoxyalkyl, Aryloxyalkyl,
Arylalkyl, Alkoxycarbonylalkyl, Carboxyalkyl; R'1 is H, Alkyl, Aryl
or Aralkyl; R2, R'2 are each the same or different and are
independently selected from H, Alkyl, Aryl or Aralkyl; T, U, V, W,
X are the same or different and may be chosen from C, N, O, S. Ru,
Rv, Rw are the same or different and may be chosen from the group
consisting in H, CN, .dbd.O, Hal, Alk, OAlk, OH, NRCN,
C(CN).dbd.C(OH)(OAlk), SR, NRR', C(O)NRR', Heterocycle, Aryl,
Heteroaryl, Cycloalkyl where Alk, Aryl, Heteroaryl, heterocycle,
Cycloalkyl are optionally substituted by Hal, NRR', CN, OH,
CF.sub.3, Aryl, Heteroaryl , OAlk. R3, R4, R5, R6 are each
identical or different and are independently chosen from the group
consisting in H, OAlk, Alk, Hal, NRR', CN, OH, OCF.sub.3, CF.sub.3,
Aryl, Heteroaryl; R and R' are each identical or different and are
independently chosen from the group consisting in H, Alk, wherein
Alk is optionally substituted by Hal, NRR', CN, OH, CF.sub.3, Aryl,
Heteroaryl; or their pharmaceutically acceptable salts, hydrates,
or hydrated salts, or the polymorphic crystalline structures of
these compounds or their optical isomers, racemates, diastereomers
or enantiomers, or their geometrical isomers (E and Z) or mixtures
thereof.
Preferably, T, U, V, W, X are C or N.
Other preferred embodiments of formula (I) are as defined above in
respect of the compounds of the invention.
Preferred compounds for the therapeutic use according to the
invention are chosen from the group consisting in:
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Methyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Butyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-decyl-oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime
[1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-ylidene]-phenyl-ami-
ne
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
lithium salt,
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts, or the polymorphic crystalline structures of these compounds
or their optical isomers, racemates, diastereomers or enantiomers,
or their regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
Most preferred compounds for the therapeutic use according to the
invention are notably selected from the group consisting in:
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-decyl-oxime 1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
lithium salt,
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts, or the polymorphic crystalline structures of these compounds
or their optical isomers, racemates, diastereomers or enantiomers,
or their regioisomers, geometrical isomers (E and Z) or mixtures
thereof.
According to a still further object, the present invention concerns
the use of a compound of formula (I), as defined above in respect
of the pharmaceutical composition, for the preparation of a
medicament for inhibiting cysteine protease.
The compounds of the invention are useful for inhibiting cysteine
proteases, in particular de-ubiquitination enzymes (such as USPs
and UCHs), caspases, cathepsins (in particular cathepsin B, D, K, S
and the like), calpains as well as viral, bacterial or parasitic
cysteine proteases in patients in the need thereof.
The compounds of the invention are particularly useful for treating
and/or preventing cancer and metastasis, more particularly prostate
and/or colon cancers, neurodegenerative diseases such as
Alzheimer's disease and Parkinson's disease, deafness, disorders
associated with ageing, inflammatory disorders, arthritis,
osteoporosis, hepatitis, liver failure, cardiac ischemia and
failure, stroke, atherosclerosis, renal failure, diabetes,
cataract; viral acute or latent infections by Herpes simplex
virus-1, Epstein-Barr virus, SARS coronavirus, rhinoviruses,
poliomyelitis virus, hepatitis A virus, hepatitis C virus,
adenoviruses, and the like; bacterial or fungal infections by
pathogenic agents belonging to the Streptococcus sp.,
Staphylococcus sp., Clostidium sp., Aspergillus sp., genera and the
like; protozoal infections by species members of the Trypanosoma
sp., Plasmodium sp., Leishmania sp., Trichomonas sp., Entamoeba
sp., Giardia sp., Toxoplasma sp., Cryptosporidium sp., genera and
the like; flat or round worm infections by species members of the
Fasciola sp., Schistosoma sp., Onchocerca sp., Ascaris sp., Taenia
sp., Caenorhabitis sp., Toxocara sp., Haemonchus sp., Ancylostoma
sp., Trichuris sp., Trichinella sp., Strongyloides sp., Brugia sp.,
genera and the like; as well as immunological, immunoregulatory or
antigen presentation disorders.
The present invention also concerns the corresponding methods of
treatment comprising the administration of a compound of the
invention together with a pharmaceutically acceptable carrier or
excipient to a patient in the need thereof.
The identification of those subjects who are in need of treatment
of herein-described diseases and conditions is well within the
ability and knowledge of one skilled in the art. A veterinarian or
a physician skilled in the art can readily identify, by the use of
clinical tests, physical examination, medical/family history or
biological and diagnostic tests, those subjects who are in need of
such treatment.
A therapeutically effective amount can be readily determined by the
attending diagnostician, as one skilled in the art, by the use of
conventional techniques and by observing results obtained under
analogous circumstances. In determining the therapeutically
effective amount, a number of factors are considered by the
attending diagnostician, including, but not limited to: the species
of subject; its size, age, and general health; the specific disease
involved; the degree of involvement or the severity of the disease;
the response of the individual subject; the particular compound
administered; the mode of administration; the bioavailability
characteristic of the preparation administered; the dose regimen
selected; the use of concomitant medication; and other relevant
circumstances.
The amount of a compound of formula (I), which is required to
achieve the desired biological effect, will vary depending upon a
number of factors, including the chemical characteristics (e.g.
hydrophobicity) of the compounds employed, the potency of the
compounds, the type of disease, the species to which the patient
belongs, the diseased state of the patient, the route of
administration, the bioavailability of the compound by the chosen
route, all factors which dictate the required dose amounts,
delivery and regimen to be administered.
"Pharmaceutically" or "pharmaceutically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate.
As used herein, "pharmaceutically acceptable excipient" includes
any carriers, diluents, adjuvants, or vehicles, such as preserving
or antioxidant agents, fillers, disintegrating agents, wetting
agents, emulsifying agents, suspending agents, solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well-known in the
art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions as suitable therapeutic
combinations.
In the context of the invention, the term "treating" or
"treatment", as used herein, means reversing, alleviating,
inhibiting the progress of, or preventing the disorder or condition
to which such term applies, or one or more symptoms of such
disorder or condition.
"Therapeutically effective amount" means an amount of a
compound/medicament according to the present invention effective in
preventing or treating a pathological condition requiring the
inhibition of an active cysteine protease involved in its
pathogenesis.
According to the invention, the term "patient", or "patient in need
thereof", is intended for an animal or a human being affected or
likely to be affected with a pathological condition involving an
active cysteine protease in its pathogenesis. Preferably, the
patient is human.
In general terms, the compounds of this invention may be provided
in an aqueous physiological buffer solution containing 0.1 to 10%
w/v compound for parenteral administration. Typical dose ranges are
from 1 .mu.g/kg to 0.1 g/kg of body weight per day; a preferred
dose range is from 0.01 mg/kg to 10 mg/kg of body weight per day or
an equivalent dose in a human child. The preferred dosage of drug
to be administered is likely to depend on such variables as the
type and extent of progression of the disease or disorder, the
overall health status of the particular patient, the relative
biological efficacy of the compound selected, the formulation of
the compound, the route of administration (intravenous,
intramuscular, or other), the pharmacokinetic properties of the
compound by the chosen delivery route, and the speed (bolus or
continuous infusion) and schedule of administrations (number of
repetitions in a given period of time).
The compounds of the present invention are also capable of being
administered in unit dose forms, wherein the term "unit dose" means
a single dose which is capable of being administered to a patient,
and which can be readily handled and packaged, remaining as a
physically and chemically stable unit dose comprising either the
active compound itself, or as a pharmaceutically acceptable
composition, as described hereinafter. As such, typical total daily
dose ranges are from 0.01 to 100 mg/kg of body weight. By way of
general guidance, unit doses for humans range from 1 mg to 3000 mg
per day. Preferably, the unit dose range is from 1 to 500 mg
administered one to six times a day, and even more preferably from
10 mg to 500 mg, once a day. Compounds provided herein can be
formulated into pharmaceutical compositions by admixture with one
or more pharmaceutically acceptable excipients. Such unit dose
compositions may be prepared for use by oral administration,
particularly in the form of tablets, simple capsules or soft gel
capsules; or intranasally, particularly in the form of powders,
nasal drops, or aerosols; or dermally, for example, topically in
ointments, creams, lotions, gels or sprays, or via trans-dermal
patches.
The compositions may conveniently be administered in unit dosage
form and may be prepared by any of the methods well-known in the
pharmaceutical art, for example, as described in Remington: The
Science and Practice of Pharmacy, 20.sup.th ed.; Gennaro, A. R.,
Ed.; Lippincott Williams & Wilkins: Philadelphia, Pa.,
2000.
Preferred formulations include pharmaceutical compositions in which
a compound of the present invention is formulated for oral or
parenteral administration.
For oral administration, tablets, pills, powders, capsules, troches
and the like can contain one or more of any of the following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, or gum tragacanth; a diluent such as
starch or lactose; a disintegrant such as starch and cellulose
derivatives; a lubricant such as magnesium stearate; a glidant such
as colloidal silicon dioxide; a sweetening agent such as sucrose or
saccharin; or a flavoring agent such as peppermint, or methyl
salicylate. Capsules can be in the form of a hard capsule or soft
capsule, which are generally made from gelatin blends optionally
blended with plasticizers, as well as a starch capsule. In
addition, dosage unit forms can contain various other materials
that modify the physical form of the dosage unit, for example,
coatings of sugar, shellac, or enteric agents. Other oral dosage
forms syrup or elixir may contain sweetening agents, preservatives,
dyes, colorings, and flavorings. In addition, the active compounds
may be incorporated into fast dissolve, modified-release or
sustained-release preparations and formulations, and wherein such
sustained-release formulations are preferably bi-modal. Preferred
tablets contain lactose, cornstarch, magnesium silicate,
croscarmellose sodium, povidone, magnesium stearate, or talc in any
combination.
Liquid preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions. The
liquid compositions may also include binders, buffers,
preservatives, chelating agents, sweetening, flavoring and coloring
agents, and the like. Non-aqueous solvents include alcohols,
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and organic esters such as ethyl oleate. Aqueous carriers
include mixtures of alcohols and water, buffered media, and saline.
In particular, biocompatible, biodegradable lactide polymer,
lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene
copolymers may be useful excipients to control the release of the
active compounds. Intravenous vehicles can include fluid and
nutrient replenishers, electrolyte replenishers, such as those
based on Ringer's dextrose, and the like. Other potentially useful
parenteral delivery systems for these active compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes.
Alternative modes of administration include formulations for
inhalation, which include such means as dry powder, aerosol, or
drops. They may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
oily solutions for administration in the form of nasal drops, or as
a gel to be applied intranasally. Formulations for buccal
administration include, for example, lozenges or pastilles and may
also include a flavored base, such as sucrose or acacia, and other
excipients such as glycocholate. Formulations suitable for rectal
administration are preferably presented as unit-dose suppositories,
with a solid based carrier, such as cocoa butter, and may include a
salicylate. Formulations for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which can be used include
petroleum jelly, lanolin, polyethylene glycols, alcohols, or their
combinations. Formulations suitable for transdermal administration
can be presented as discrete patches and can be lipophilic
emulsions or buffered, aqueous solutions, dissolved and/or
dispersed in a polymer or an adhesive.
The invention is further illustrated but not restricted by the
description in the following examples.
Representative compounds of the invention are summarized in the
table below:
TABLE-US-00001 Preparation CHEMISTRY Procedure ##STR00015## Ex 5a/E
##STR00016## Ex 5b/E ##STR00017## Ex 5c/E ##STR00018## Ex 5d/E
##STR00019## Ex 5e/E ##STR00020## Ex 6 ##STR00021## Ex 7
##STR00022## Ex 8a/F ##STR00023## Ex 8b/F ##STR00024## Ex 8c/F
##STR00025## Ex 10a/K ##STR00026## Ex 10b/K ##STR00027## Ex 10c/K
##STR00028## Ex 10d/K ##STR00029## Ex 10e/K ##STR00030## Ex 10f/K
##STR00031## Ex 10g/K ##STR00032## Ex 10h/K ##STR00033## Ex 11
##STR00034## Ex 12 ##STR00035## Ex 13
EXPERIMENTAL
Representative compounds of the invention can be synthesized
according to the following procedures:
General Procedure A: Synthesis of
pentaaza-cyclopenta[b]fluoren-9-one:
##STR00036##
A mixture of R1-substituted (1,2,4)-triazole-3,4-diamine (8.8 mmol)
and ninhydrin (1.57 g, 8.8 mmol) in EtOH (10 ml) and AcOH (1.5 ml)
was refluxed for 2-16 hours. The solvent was removed under reduced
pressure and the residue was dissolved in EtOAc, washed with
saturated K.sub.2CO.sub.3 and brine. The organic phase was dried
over Na.sub.2SO.sub.4, filtered and the solvents removed by
evaporation under reduced pressure. The crude was purified as
follows: silica gel flash chromatography (toluene/MeOH 95:5 to 8:2
or CH.sub.2Cl.sub.2/EtOAc 9:1 to 1:1) for the purification of the
regioisomeric mixture, then neutral alumina (grade II) flash
chromatography (CH.sub.2Cl.sub.2/EtOAc 7:3 to CH.sub.2Cl.sub.2/MeOH
1:1+5% HCOOH or AcOH) for the separation of the regioisomers.
1-Methyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
(1b/A)
Prepared according to the general procedure A in 13% yield as
yellow solid. .sup.1H NMR (300 MHz, DMSO d.sub.6): .delta. 8.23 (d,
1H), 8.02 (m, 2H), 7.89 (ddd, 1H), 2.72 (s, 3H). ESI.sup.+MS: calcd
for C.sub.12H.sub.7N.sub.5O: 237.22; found: 238.2 (MH.sup.+).
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one (1b/B)
Prepared according to the general procedure A in 30% yield as
yellow solid. .sup.1H NMR (300 MHz, DMSO d.sub.6): .delta. 8.16 (d,
1H), 8.05-7.95 (m, 2H), 7.85 (ddd, 1H), 2.77 (s, 3H). ESI.sup.+MS:
calcd for C.sub.12H.sub.7N.sub.5O: 237.22; found: 238.2
(MH.sup.+).
General Procedure B: Synthesis of
pentaaza-cyclopenta[b]fluoren-9-one:
##STR00037##
The preparation of diaminotriazoles follows the procedure reported
in Eur. J. Med. Chem.-Chim. Ther. 1986, 21, 235.
A mixture of diaminoguanidine hydrochloride (1 g, 8 mmol) in an
excess (10 g) of the appropriate carboxylic acid was stirred and
heated at 120-130.degree. C. for 12-24 hours. The solution was
cooled to room temperature and HCl 37% (10 ml) was added. The
mixture was refluxed for 2-3 hours and then concentrated to dryness
in vacuo. The obtained crude was washed with Et.sub.2O (.times.3)
and used without any further purification.
For the condensation between the crude diaminotriazole and
ninhydrin, see the General procedure A.
1-Butyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one (1f/A)
Prepared according to the general procedure B in 6% yield as yellow
solid. .sup.1H NMR (300 MHz, DMSO d.sub.6): .delta. 8.23 (d, 1H),
8.02 (m, 2H), 7.89 (ddd, 1H), 3.10 (dd, 2H), 1.81 (m, 2H), 1.42 (m,
2H), 0.94 (t, 3H). ESI.sup.+MS: calcd for C.sub.15H.sub.13N.sub.5O:
279.30; found: 280.2 (MH.sup.+).
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one (1f/B)
Prepared according to the general procedure B in 10% yield as
yellow solid. .sup.1H NMR (300 MHz, DMSO d.sub.6): .delta. 8.16
(d,1H), 7.99 (m, 2H), 7.85 (dd,1H), 3.16 (dd, 2H), 1.87 (m, 2H),
1.44 (m, 2H), 0.96 (t, 3H). ESI.sup.+MS: calcd for
C.sub.15H.sub.13N.sub.5O: 279.30; found: 280.3 (MH.sup.+).
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one (1g/A)
Prepared according to the general procedure B in 48% yield as
yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.21 (d,
1H), 8.00 (d, 1H), 7.90 (ddd, 1H), 7.77(ddd, 1H), 3.21 (q, 2H),
1.49 (t, 3H). ESI.sup.+MS: calcd for C.sub.13H.sub.9N.sub.5O:
251.25; found: 252.1 (MH.sup.+).
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one (1g/B)
Prepared according to the general procedure B in 32% yield as
yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.12 (d,
1H), 8.02 (d, 1H), 7.88 (ddd, 1H), 7.75 (ddd, 1H), 3.25 (q, 2H),
1.53 (t, 3H). ESI.sup.+MS: calcd for C.sub.13H.sub.9N.sub.5O:
251.25; found: 252.1 (MH.sup.+).
General Procedure E: Synthesis of O-alkyloxime derivatives of
pentaaza-cyclopenta [b]fluoren-9-one:
##STR00038##
A suspension of 1 (1 mmol), O-alkyl-hydroxylamine hydrochloride (3
mmol) and molecular sieves in pyridine (10 ml) was heated to
60.degree. C. for 2-12 h. The insoluble residue was filtered, the
solvent evaporated and the crude purified by flash chromatography
on silica gel (CH.sub.2Cl.sub.2/acetone 85:15 or toluene/MeOH 9:1
or petroleum spirit/EtOAc 1:1).
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime (5a)
Prepared according to the general procedure E from 1b/B in 55%
yield as yellow solid as 2:1 E/Z mixture. .sup.1H NMR (300 MHz,
DMSO d.sub.6) (mixture of isomers): .delta. 8.43 (m, 1H), 8.16 (m,
1H), 7.81 (m, 2H), 4.34 (s, 3H), 2.75 (s, 3H). 8.05 (m, 1H), 7.92
(m, 1H), 7.72 (m, 2H), 4.30 (s, 3H), 2.75 (s, 3H). ESI.sup.+MS:
calcd for C.sub.13H.sub.10N.sub.6O: 266.26; found: 267.1
(MH.sup.+).
3-Methyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (5b)
Prepared according to the general procedure E from 1b/B in 65%
yield as yellow solid as 1:1 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3) (mixture of isomers): .delta. 8.02 (d, 1H), 7.95 (d,
1H), 7.75-7.56 (m, 2H), 6.26-6.08 (m, 1H), 5.50 (dd, 1H), 5.35 (d,
1H), 5.05 (d, 2H), 2.86 (s, 3H). 8.49 (m, 1H), 8.13 (m, 1H),
7.77-7.56 (m, 2H), 6.26-6.08 (m, 1H), 5.50 (dd, 1H), 5.39 (d, 1H),
5.12 (d, 2H), 2.86 (s, 3H). ESI.sup.+MS: calcd for
C.sub.15H.sub.12N.sub.6O: 292.30; found: 293.1 (MH.sup.+).
1-Methyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (5c)
Prepared according to the general procedure E from 1b/A in 76%
yield as yellow solid as 7:3 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3) (mixture of isomers): .delta. 8.16 (m, 1H), 7.95 (m,
1H), 7.77-7.60 (m, 2H), 6.26-6.08 (m, 1H), 5.54 (ddt, 1H), 5.37
(ddt, 1H), 5.04 (ddd, 2H), 2.84 (s, 3H). 8.49 (m, 1H), 8.26 (m,
1H), 7.77-7.60 (m, 2H), 6.26-6.08 (m, 1H), 5.49 (ddt, 1H), 5.40
(ddt, 1H), 5.09 (ddd, 2H), 2.88 (s, 3H). ESI.sup.+MS: calcd for
C.sub.15H.sub.12N.sub.6O: 292.30; found: 293.1 (MH.sup.+).
3-Butyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (5d)
Prepared according to the general procedure E from 1f/B in 93%
yield as yellow solid as 6:4 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3) (mixture of isomers): .delta. 8.42 (m, 1H), 8.06 (m,
1H), 7.64 (m, 2H), 6.19-6.00 (m, 1H), 5.41 (m, 1H), 5.31 (m, 1H),
5.03 (ddd, 2H), 3.17 (dd, 2H), 1.88 (m, 2H), 1.43 (m, 2H), 0.93 (t,
3H). 7.96 (m, 1H), 7.87 (m, 1H), 7.55 (m, 2H), 6.19-6.00 (m, 1H),
5.41 (m, 1H), 5.26 (m, 1H), 4.97 (ddd, 2H), 3.17 (dd, 2H), 1.88 (m,
2H), 1.43 (m, 2H), 0.93 (t, 3H). ESI.sup.+MS: calcd for
C.sub.18H.sub.18N.sub.6O: 334.38; found: 335.1 (MH.sup.+).
1-Butyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (5e)
Prepared according to the general procedure E from 1f/A in 95%
yield as yellow solid as 1:1 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3) (mixture of isomers): .delta. 8.45 (m, 1H), 8.22 (m,
1H), 7.69 (m, 2H), 6.22-6.02 (m, 1H), 5.45 (m, 1H), 5.35 (m, 1H),
5.05 (ddd, 2H), 3.21 (dd, 2H), 1.91 (m, 2H), 1.45 (m, 2H), 0.95 (t,
3H). 8.12 (m, 1H), 7.91 (m, 1H), 7.62 (m, 2H), 6.22-6.02 (m, 1H),
5.49 (m, 1H), 5.32 (m, 1H), 4.99 (ddd, 2H), 3.18 (dd, 2H), 1.91 (m,
2H), 1.45 (m, 2H), 0.95 (t, 3H). ESI.sup.+MS: calcd for
C.sub.18H.sub.18N.sub.6O: 334.38; found: 335.2 (MH.sup.+).
Synthesis of 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime and/or its Corresponding Regioisomeric tetrazol
(6):
##STR00039##
The compound was prepared according to the general procedure E from
a 6:4 regioisomeric mixture of
1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one (prepared from
ninhydrin and tetrazole-1,5-diamine) in 89% yield as yellow solid
as E/Z and regioisomeric mixture. .sup.1H NMR (300 MHz, CDCl.sub.3)
(mixture of isomers): .delta. 8.47 (m, 1H), 8.22 (m, 1H), 7.84-7.58
(m, 2H), 6.23-6.03 (m, 1H), 5.46 (m, 1H), 5.37 (m, 1H), 5.13 (ddd,
2H). 8.19 (m, 1H), 7.98 (m, 1H), 7.84-7.58 (m, 2H), 6.23-6.03 (m,
1H), 5.46 (m, 1H), 5.34 (m, 1H), 5.06 (m, 2H). ESI.sup.+MS: calcd
for C.sub.13H.sub.9N.sub.7O: 279.26; found: 280.2 (MH.sup.+).
Synthesis of 1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one oxime
and/or its Corresponding Regioisomeric tetrazol (7):
##STR00040##
The compound was prepared according to the general procedure E from
a 6:4 regioisomeric mixture of
1,2,3,3a,4,10-hexaaza-cyclopenta[b]fluoren-9-one (prepared from
ninhydrin and tetrazole-1,5-diamine) in 44% yield as yellow solid
as E/Z and regioisomeric mixture. .sup.1H NMR (300 MHz, CDCl.sub.3)
(mixture of isomers): .delta. 13.87 (bs, 1H), 8.59 (m, 1H), 8.14
(m, 1H), 7.78-7.52 (m, 2H). 13.69 (bs, 1H), 8.05 (d, 1H), 7.91 (d,
1H), 7.78-7.52 (m, 2H). ESI.sup.+MS: calcd for
C.sub.10H.sub.5N.sub.7O: 239.20; found: 240.1 (MH.sup.+).
General Procedure F: Synthesis of O-alkyloxime of
hexaaza-cyclopenta[b]fluoren-9-one:
##STR00041##
A mixture of 7 (48 mg, 0.20 mmol), alkyl bromide (0.6 mmol) and
K.sub.2CO.sub.3 (55 mg, 0.4 mmol) in DMF (2 ml) was stirred at room
temperature for 16 h, then the solvent was evaporated under reduced
pressure. The crude was purified by flash chromatography
(CH.sub.2Cl.sub.2 in variable mixture with MeOH or petroleum
ether).
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one O-decyl-oxime
and/or its Corresponding Regioisomeric tetrazol (8a)
Prepared according to the general procedure F in 53% yield as
yellow-green solid as E/Z and regioisomeric mixture. .sup.1H NMR
(300 MHz, CDCl.sub.3) (mixture of isomers): .delta. 8.39 (m, 1H),
8.24 and 8.15 (m, 1H), 7.78-7.63 (m, 2H), 4.61-4.47 (m, 2H), 1.82
(m, 2H), 1.47-1.06 (m, 14H), 0.75 (m, 3H). ESI.sup.+MS: calcd for
C.sub.20H.sub.25N.sub.7O: 379.47; found: 380.2 (MH.sup.+).
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(2-methoxy-ethyl)-oxime and/or its Corresponding Regioisomeric
tetrazol_(8b)
Prepared according to the general procedure F in 29% yield as light
brown solid as E/Z and regioisomeric mixture. .sup.1H NMR (300 MHz,
DMSO d.sub.6) (mixture of isomers): .delta. 8.49 (m, 1H), 8.27 (m,
1H), 7.83-7.66 (m, 2H), 4.73 (m, 2H), 3.82 (m, 2H), 3.40 (s, 3H).
8.49 (m, 1H), 8.19 (m, 1H), 7.83-7.66 (m, 2H), 4.73 (m, 2H), 3.82
(m, 2H), 3.41 (s, 3H). ESI.sup.+MS: calcd for
C.sub.13H.sub.11N.sub.7O.sub.2: 297.28; found 298.0 (MH.sup.+).
1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-one
O-(3-phenoxy-propyl)-oxime and/or its Corresponding Regioisomeric
tetrazol (8c)
Prepared according to the general procedure F in 42% yield as
yellow solid as E/Z and regioisomeric mixture. .sup.1H NMR (300
MHz, CDCl.sub.3) (mixture of isomers): .delta. 8.41 (m, 1H), 8.15
(m, 1H), 7.76-7.58 (m, 2H), 7.18 (m, 2H), 6.83 (m, 3H), 4.87-4.70
(m, 2H), 4.18-4.07 (m, 2H), 2.42-2.27 (m, 2H). 8.26 (m, 1H), 7.89
(d, 1H), 7.76-7.58 (m, 2H), 7.18 (m, 2H), 6.83 (m, 3H), 4.87-4.70
(m, 2H), 4.18-4.07 (m, 2H), 2.42-2.27 (m, 2H). ESI.sup.+MS: calcd
for C.sub.19H.sub.15N.sub.7O.sub.2: 373.38; found: 374.1
(MH.sup.+).
General Procedure K: Synthesis of O-alkyloxime Derivatives of ethyl
pentaaza-cyclopenta[b]fluoren-9-one:
##STR00042##
A suspension of 1g/A or 1g/B (1 mmol), O-alkyl-hydroxylamine
hydrochloride (2 mmol) and molecular sieves in pyridine (10 ml) was
heated to 60.degree. C. for 2-3 h. The insoluble residue was
filtered, the solvent evaporated and the crude purified by flash
chromatography on silica gel.
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime (10a)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:17:3) from 1g/A in quantitative
yield as yellow solid as 7:3 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.47 (m, 1H), 8.27 (m, 1H), 7.73 (m, 1H), 7.66
(m, 1H), 4.41 (s, 3H), 3.28 (q, 2H), 1.55 (t, 3H) and 8.17 (m, 1H),
7.96 (m, 1H), 7.73 (m, 1H), 7.63 (m, 1H), 4.37 (s, 3H), 3.25 (q,
2H), 1.55 (t, 3H). ESI.sup.+MS: calcd for C.sub.14H.sub.12N.sub.6O:
280.29; found: 281.1 (MH.sup.+).
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-methyl-oxime (10b)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:17:3) from 1g/B in quantitative
yield as yellow solid as 7:3 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.50 (m, 1H), 8.17 (m, 1H), 7.63 (m, 2H), 4.45
(s, 3H), 3.30 (q, 2H), 1.57 (t, 3H) and 8.07 (d, 1H), 7.98 (d, 1H),
7.68 (ddd, 1H), 7.64 (ddd, 1H), 4.41 (s, 3H), 3.29 (q, 2H), 1.59
(t, 3H). ESI.sup.+MS: calcd for C.sub.14H.sub.12N.sub.6O: 280.29;
found: 281.1 (MH.sup.+).
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime (10c)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 70:25:5) from 1g/A in quantitative
yield as yellow solid as 6:4 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.17 (m, 1H), 7.96 (m, 1H), 7.73 (m, 1H), 7.65
(ddd, 1H), 4.60 (q, 2H), 3.26 (q, 2H), 1.55 (t, 3H), 1.55 (t, 3H)
and 8.47 (m, 1H), 8.27 (m, 1H); 7.72 (m, 1H), 7.63 (ddd, 1H), 4.66
(q, 2H), 3.30 (q, 2H), 1.54 (t, 3), 1.51 (t, 3H). ESI.sup.+MS:
calcd for C.sub.15H.sub.14N.sub.6O: 294.32; found: 295.1
(MH.sup.+).
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-ethyl-oxime (10d)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 70:25:5) from 1g/B in quantitative
yield as yellow solid as 1:1 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.49 (m, 1H), 8.13 (m, 1H), 7.70 (m, 1H), 7.62
(m, 1H), 4.69 (q, 2H), 3.27 (q, 2H), 1.58-1.48 (m, 6H), and 8.03
(m, 1H), 7.96 (m, 1H), 7.70 (m, 1H), 7.62 (m, 1H), 4.62 (q, 2H),
3.27 (q, 2H), 1.58-1.48 (m, ESI.sup.+MS: calcd for
C.sub.15H.sub.14N.sub.6O: 294.32; found: 295.1 (MH.sup.+).
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (10e)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:16:4) from 1g/A in quantitative
yield as yellow solid as 6:4 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.17 (d, 1H), 7.95 (d, 1H), 7.65 (m, 2H),
6.26-6.07 (m, 1H), 5.54 (m, 1H), 5.37 (m, 1H), 5.04 (ddd, 2H), 3.26
(m, 2H), 1.54 (m, 3H) and 8.49 (d, 1H), 8.27 (d, 1H), 7.73 (m, 2H),
6.26-6.07 (m, 1H), 5.49 (m, 1H), 5.40 (m, 1H), 5.09 (ddd, 2H), 3.26
(m, 2H), 1.54 (m, 3H). ESI.sup.+MS: calcd for
C.sub.16H.sub.14N.sub.6O: 306.33; found: 307.1 (MH.sup.+).
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-allyl-oxime (10f)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:17:3) from 1g/B in 96% yield as
yellow solid as 65:35 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.50 (m, 1H), 8.14 (m, 1H), 7.71 (m, 1H), 7.65
(m, 1H), 6.26-6.09 (m, 1H), 5.53 (m, 1H), 5.39 (m, 1H), 5.12 (ddd,
2H), 3.27 (q, 2H), 1.55 (t, 3H) and 8.04 (m, 1H), 7.95 (m, 1H),
7.71 (m, 1H), 7.61 (m, 1H), 6.26-6.09 (m, 1H), 5.47 (m, 1H), 5.36
(m, 1H), 5.106 (ddd, 2H), 3.27 (q, 2H), 1.56 (t, 3H). ESI.sup.+MS:
calcd for C.sub.16H.sub.14N.sub.6O: 306.33; found: 307.2
(MH.sup.+).
1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime (10g)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:17:3) from 1g/A in 86% yield as
yellow solid as 65:35 E/Z mixture. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.16 (m, 1H), 7.96 (m, 1H), 7.70 (m, 1H), 7.65
(m, 1H), 7.52 (m, 2H), 7.41 (m, 3H), 5.58 (s, 2H), 3.21 (q, 2H),
1.49 (t, 3H) and 8.43 (m, 1H), 8.26 (m, 1H), 7.70 (m, 1H), 7.65 (m,
1H), 7.52 (m, 2H), 7.41 (m, 3H), H), 5.62 (s, 2H), 3.29 (q, 2H),
1.56 (t, 3H). ESI.sup.+MS: calcd for C.sub.20H.sub.16N.sub.6O:
356.39; found: 357.1 (MH.sup.+).
3-Ethyl-1,2,3a,4,10-pentaaza-cyclopenta[b]fluoren-9-one
O-benzyl-oxime (10h)
Prepared according to the general procedure K (eluent:
CH.sub.2Cl.sub.2/EtOAc/MeOH 80:17:3) from 1g/B in 99% yield as
yellow solid as 6:4 E/Z mixture. .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 8.44 (m, 1H), 8.13 (m, 1H), 7.67 (m, 1H), 7.61 (m, 1H),
7.52 (m, 2H), 7.46-7.29 (m, 3H), 5.64 (s, 2H), 3.62 (q, 2H), 1.55
(t, 3H) and 8.01 (m, 1H), 7.92 (m, 1H), 7.70 (m, 1H), 7.65 (m, 1H),
7.52 (m, 2H), 7.41 (m, 3H), 5.59 (s, 2H), 3.29 (q, 2H), 1.56 (t,
3H). ESI.sup.+MS: calcd for C.sub.20H.sub.16N.sub.6O: 356.39;
found: 357.1 (MH.sup.+).
Synthesis of
[1-Ethyl-2,3,4,10,10a-pentaaza-cyclopenta[b]fluoren-9-ylidene]-phenyl-ami-
ne (11):
##STR00043##
To a suspension of 1g/A (200 mg, 0.79 mmol) and molecular sieves in
toluene (4 ml), aniline (72 .mu.l, 0.79 mmol) was added. The
mixture was stirred at 130.degree. C. for 4 h, then the solvent was
evaporated and he crude purified by flash chromatography
(CH.sub.2Cl.sub.2/EtOAc/MeOH 80:18:2), affording 11 (231 mg, 90%)
as orange solid in diastereoisomeric ratio 1:1.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.28 (d, 1H), 7.70 (ddd,
1H), 7.56-7.26 (m, 6H), 6.91 (d, 1H), 3.34 (q, 2H), 1.58 (t, 3H)
and 8.22 (m, 2H), 7.81 (m, 2H), 7.47 (m, 1H), 7.07 (m, 4H), 2.80
(q, 2H), 1.21 (t, 3H). ESI+MS: calcd for C.sub.19H.sub.14N.sub.6:
326.36; found: 327.2 (MH.sup.+).
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetic
acid ethyl ester and/or its Corresponding Regioisomeric tetrazol
(12)
##STR00044##
A mixture of oxime 7 (560 mg, 2.34 mmol) and cesium carbonate (1.54
g, 4.68 mmol) were stirred in DMF (12 ml) for 5 min. Ethyl
bromoacetate (1.2 g, 7.02 mmol) was added dropwise, and at the end
of the addition, the deeply colored mixture was stirred for 3 h at
room temperature. The solvent was evaporated, and the crude product
dissolved in dichloromethane. After filtration over a pad of
silice, evaporation, recrystallisation with cyclohexane/ethyl
acetate and trituration with cyclohexane, 717 mg (94%) of compound
12 was obtained as a green powder.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO) (mixture of isomers):
.delta.(ppm)=1.28 (m, 3H); 4.21 (m, 2H); 5.28 (m, 2H); 7.70-8.60
(m, 4H). LC-MS (ES): m/z=651 (2M+H.sup.+), 326 (M+H.sup.+).
(1,2,3,3a,4,10-Hexaaza-cyclopenta[b]fluoren-9-ylideneaminooxy)-acetate
Lithium salt and/or its Corresponding Regioisomeric tetrazol
(13)
##STR00045##
A solution of ester 12 (700 mg; 2.15 mmol) and LiOH (451 mg, 10.75
mmol) in 12 ml methanol were stirred for 2 h at room temperature.
The deeply coloured mixture was cooled to -20.degree. C., and after
1 h, the predpitate formed filtered and washed with cold methanol
to leave 380 mg (59%) of compound 13 as a green solid.
.sup.1H-NMR (400 MHz, D.sub.2O) (mixture of isomers):
.delta.(ppm)=4.8 (s, 2H); 7.40-8.40 (m, 4H). LC-MS (ES): m/z=296
(M-H.sup.+).
Representative Cysteine Proteases
USP5 Activity Assay
USP5 was diluted in USP buffer (50 mM Tris HCl; 0.5 mM EDTA; 5 mM
DTT; 0.01% Triton X-100; Bovine Serum Albumin 0.05 mg.ml.sup.-1
pH7.6). Compounds stocks (100 mM) were stored at -20.degree. C. in
DMSO. Compounds were tested at the following final concentrations:
100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M; 1.23 .mu.M; 412 nM;
137 nM; 45.7 nM; 15.2 nM; 5 nM.
Reactions were performed as duplicates in Black LJL 96 well plates
(HE microplates; Molecular Devices; 20 .mu.l final reaction
volume). The substrate concentration for USP5 was 400 nM Ub-AMC
(Boston Biochem). The concentrations of the enzyme (USP5) in
specificity assays was 300 pM. The concentrations were determined
in order to perform specificity assays under initial velocities at
fixed substrate concentration. Compounds were pre-incubated with
enzymes for 30 minutes at 25.degree. C. Reactions were initiated by
addition of substrate to the plates containing the enzymes
(+/-compounds) diluted in assay buffer. Reactions were incubated
for 60 minutes at 37.degree. C. Reactions were stopped by adding
acetic acid (100 mM final). Readings were performed on a Pherastar
Fluorescent Reader (BMG). .lamda. Emission 380 nm; .lamda.
Excitation=460 nm. Data (mean values+/-standard deviation) were
analyzed as % of control (no compound) and plotted as percentage
versus the Log of the compound concentration using GraphPad
(Prism). Data were fitted to a sigmoidal model (variable
slope).
Cloning & Purification of USP7
The cDNA encoding USP7 was obtained by PCR amplification from
placenta mRNA. USP7 cDNA was subcloned by PCR into a baculovirus
expression vector (pFastBac-HT; Invitrogen). A cDNA encoding a
mutated USP7 was generated by mutagenic PCR. The corresponding
protein encodes a cysteine to alanine substitution at residue 223.
The sequences were ascertained by sequencing of the entire open
reading frame. Bacmids encoding USP7 were generated following
DH10bac transposition. The corresponding bacmids were transfected
into insect cells (Sf9). Viruses were recovered from culture
supernatant and amplified twice. Insect cells (Sf9 or High Five;
Invitrogen) were infected for 72 hours. Total cell lysates were
harvested and lyzed in lysis buffer (Tris HCl 50 mM pH7.6; 0.75%
NP40; 500 mM NaCl; 10% glycerol; 1 mM DTT; 10 mM imidazole;
Protease Inhibitor Cocktail; AEBSF 20 .mu.g.ml.sup.-1; Aprotinin 10
.mu.g.ml.sup.-1). Proteins were affinity purified on metal affinity
resins (Talon Metal affinity resin; BD Biosciences). Bound
materials were extensively washed in wash buffer (50 mM Sodium
Phosphate pH7.0; 300 mM NaCl; 10 mM imidazole; 0.5% Triton X-100;
10% glycerol) and eluted from the resin in 250 mM
imidazole-containing wash buffer. Proteins were dialyzed in
dialysis buffer (Tris HCl pH 7.6 20 mM; NaCl 200 mM; DTT 1 mM; EDTA
1 mM; 10% Glycerol). Proteins purifications were analyzed on 4-12%
NuPAGE (Invitrogen).
USP7 Activity Assay
USP7 was diluted in USP buffer (50 mM Tris HCl; 0.5 mM EDTA; 5 mM
DTT; 0.01% Triton X-100; Bovine Serum Albumin 0.05 mg.ml.sup.-1
pH7.6). Compounds stocks (100 mM) were stored at -20.degree. C. in
DMSO. Compounds were tested at the following final concentrations:
100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M; 1.23 .mu.M; 412 nM;
137 nM; 45.7 nM; 15.2 nM; 5 nM.
Reactions were performed as duplicates in Black LJL 96 well plates
(HE microplates; Molecular Devices; 20 .mu.l final reaction
volume). The substrate concentration for USP7 was 400 nM Ub-AMC
(Chem. Biol., 2003, 10, p. 837-846) (Boston Biochem). The
concentrations of the enzyme (USP7) in specificity assays was 152
pM. The concentrations were determined in order to perform
specificity assays under initial velocities at fixed substrate
concentration. Compounds were pre-incubated with enzymes for 30
minutes at 25.degree. C. Reactions were initiated by addition of
substrate to the plates containing the enzymes (+/-compounds)
diluted in assay buffer. Reactions were incubated for 60 minutes at
37.degree. C. Reactions were stopped by adding acetic acid (100 mM
final). Readings were performed on a Pherastar Fluorescent Reader
(BMG). .lamda. Emission 380 nm; .lamda. Excitation=460 nm. Data
(mean values+/-standard deviation) were analyzed as % of control
(no compound) and plotted as percentage versus the Log of the
compound concentration using GraphPad (Prism). Data were fitted to
a sigmoidal model (variable slope).
Cloning & Purification of USP8
The cDNA encoding USP8 was obtained by PCR amplification from
placenta mRNA. USP8 cDNA was subcloned by PCR into a baculovirus
expression vector (pFastBac-HT; Invitrogen). A cDNA encoding a
mutated USP8 was generated by mutagenic PCR. The corresponding
protein encodes a cysteine to alanine substitution at residue 786.
The sequences were ascertained by sequencing of the entire open
reading frame. Bacmids encoding USP7 were generated following
DH10bac transposition. The corresponding bacmids were transfected
into insect cells (Sf9). Viruses were recovered from culture
supernatant and amplified twice. Insect cells (Sf9 or High Five;
Invitrogen) were infected for 72 hours. Total cell lysates were
harvested and lyzed in lysis buffer (Tris HCl 50 mM pH7.6; 0.75%
NP40; 500 mM NaCl; 10% glycerol; 1 mM DTT; 10 mM imidazole;
Protease Inhibitor Cocktail; AEBSF 20 .mu.g.ml.sup.-1; Aprotinin 10
.mu.g.ml.sup.-1). Proteins were affinity purified on metal affinity
resins (Talon Metal affinity resin; BD Biosciences). Bound
materials were extensively washed in wash buffer (50 mM Sodium
Phosphate pH 7.0; 300 mM NaCl; 10 mM imidazole; 0.5% Triton X-100;
10% glycerol) and eluted from the resin in 250 mM
imidazole-containing wash buffer. Proteins were dialyzed in
dialysis buffer (Tris HCl pH 7.6 20 mM; NaCl 200 mM; DTT 1 mM; EDTA
1 mM; 10% Glycerol). Proteins purifications were analyzed on 4-12%
NuPAGE (Invitrogen).
USP8 Activity Assay
USP8 was diluted in USP buffer (50 mM Tris HCl; 0.5 mM EDTA; 5 mM
DTT; 0.01% Triton X-100; Bovine Serum Albumin 0.05 mg.ml.sup.-1
pH8.8). Compounds stocks (100 mM) were stored at -20.degree. C. in
DMSO. Compounds were tested at the following final concentrations:
100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M; 1.23 .mu.M; 412 nM;
137 nM; 45.7 nM; 15.2 nM; 5 nM.
Reactions were performed as duplicates in Black LJL 96 well plates
(HE microplates; Molecular Devices; 20 .mu.l final reaction
volume).The substrate concentration for USP8 was 400 nM Ub-AMC
(Boston Biochem). The concentrations of the enzyme (USP8) in
specificity assays was 630 pM. The concentrations were determined
in order to perform specificity assays under initial velocities at
fixed substrate concentration. Compounds were pre-incubated with
enzymes for 30 minutes at 25.degree. C. Reactions were initiated by
addition of substrate to the plates containing the enzymes
(+/-compounds) diluted in assay buffer. Reactions were incubated
for 60 minutes at 37.degree. C. Reactions were stopped by adding
acetic acid (100 mM final). Readings were performed on a Pherastar
Fluorescent Reader (BMG). .lamda. Emission 380 nm; .lamda.
Excitation=460 nm. Data (mean values.+-.standard deviation) were
analyzed as % of control (no compound) and plotted as percentage
versus the Log of the compound concentration using GraphPad
(Prism). Data were fitted to a sigmoidal model (variable
slope).
UCH-L3 Activity Assay
Uch-L3 was diluted in USP buffer (50 mM Tris HCl; 0.5 mM EDTA; 5 mM
DTT; 0.01% Triton X-100; Bovine Serum Albumin 0.05 mg.ml.sup.-1
pH7.6). Compounds stocks (100 mM) were stored at -20.degree. C. in
DMSO. Compounds were tested at the following final concentrations:
100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M; 1.23 .mu.M; 412 nM;
137 nM; 45.7 nM; 15.2 nM; 5 nM.
Reactions were performed as duplicates in Black LJL 96 well plates
(HE microplates; Molecular Devices; 20 .mu.l final reaction
volume).The substrate concentration for Uch-L3 was 400 nM Ub-AMC
(Boston Biochem). The concentration of the enzyme (Uch-L3) in
specificity assays was 13 pM. The concentrations were determined in
order to perform specificity assays under initial velocities at
fixed substrate concentration. Compounds were pre-incubated with
enzymes for 30 minutes at 25.degree. C. Reactions were initiated by
addition of substrate to the plates containing the enzymes
(+/-compounds) diluted in assay buffer. Reactions were incubated
for 60 minutes at 37.degree. C. Reactions were stopped by adding
acetic acid (100 mM final). Readings were performed on a Pherastar
Fluorescent Reader (BMG). .DELTA. Emission 380 nm; .delta.
Excitation=460 nm. Data (mean values+/-standard deviation) were
analyzed as % of control (no compound) and plotted as percentage
versus the Log of the compound concentration using GraphPad
(Prism). Data were fitted to a sigmoidal model (variable
slope).
Caspase 3 Activity Assay
Caspase 3 was diluted in Caspase 3 buffer (100 mM Hepes pH 7.5; 10%
sucrose; 0.1% CHAPS). Compounds stocks (100 mM) were stored at
-20.degree. C. in DMSO. Compounds were tested at the following
final concentrations: 100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M;
1.23 .mu.M; 412 nM; 137 nM; 45.7 nM; 15.2 nM; 5 nM. Reactions were
performed as duplicates in Black LJL 96 well plates (HE
microplates; Molecular Devices; 20 .mu.l final reaction volume).
The substrate concentration for caspase 3 specificity assay was 500
nM (Ac-DEVD-AMC; Promega). The concentration of the enzyme (Caspase
3) in specificity assays was 3.2 nM. The concentrations were
determined in order to perform specificity assays under initial
velocities at fixed substrate concentration. Compounds were
pre-incubated with enzymes for 30 minutes at 25.degree. C.
Reactions were initiated by addition of substrate to the plates
containing the enzymes (+/-compounds) diluted in assay buffer.
Reactions were incubated for 60 minutes at 37.degree. C. Reactions
were stopped by adding acetic acid(100 mM final). Readings were
performed on a Pherastar Fluorescent Reader (BMG). .delta. Emission
380 nm; .delta. Excitation=460 nm. Data (mean values+/-standard
deviation) were analyzed as % of control (no compound) and plotted
as percentage versus the Log of the compound concentration using
GraphPad (Prism). Data were fitted to a sigmoidal model (variable
slope).
Cathepsin B Activity Assay
Cathepsin B was diluted in Cathepsin B buffer (20 mM Tris HCl pH
6.8; 1 mM EDTA; 1 mM DTT). Compounds stocks (100 mM) were stored at
-20.degree. C. in DMSO. Compounds were tested at the following
final concentrations: 100 .mu.M; 33.3 .mu.M; 11.1 .mu.M; 3.7 .mu.M;
1.23 .mu.M; 412 nM; 137 nM; 45.7 nM; 15.2 nM; 5 nM. Reactions were
performed as duplicates in Black LJL 96 well plates (HE
microplates; Molecular Devices; 20 .mu.l final reaction volume).
The substrate concentration for cathepsin B specificity assay was
36 .mu.M (z-RR-AMC; Calbiochem).The concentration of the enzyme
(Cathepsin B) in specificity assays was 3.6 nM. The concentrations
were determined in order to perform specificity assays under
initial velocities at fixed substrate concentration. Compounds were
pre-incubated with enzymes for 30 minutes at 25.degree. C.
Reactions were initiated by addition of substrate to the plates
containing the enzymes (+/-compounds) diluted in assay buffer.
Reactions were incubated for 60 minutes at 37.degree. C. Reactions
were stopped by adding acetic acid (100 mM final). Readings were
performed on a Pherastar Fluorescent Reader (BMG). .delta. Emission
380 nm; .delta. Excitation=460 nm. Data (mean values+/-standard
deviation) were analyzed as % of control (no compound) and plotted
as percentage versus the Log of the compound concentration using
GraphPad (Prism). Data were fitted to a sigmoidal model (variable
slope).
Cell Viability and Proliferation Methods
HCT116 Cell Viability and Proliferation Assay
HCT116 colon cancer cells were obtained from ATCC (American Type
Culture Collection), and maintained in Mc Coy's 5A medium
containing 10% FBS, 3 mM glutamine and 1% penicillin/streptomycin.
Cells were incubated at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2.
Cell viability was assayed using the MTS technique in 96-well
culture plates (CellTiter 96.RTM. Aqueous Non-Radioactive Cell
Proliferation Assay, Promega) according to the manufacturer's
instructions. MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium) is a MTT-derived tetrazolium that is reduced in
metabolically active cells into a soluble, cell-permeant formazan.
The amount of formazan, detected by its absorbance at 492 nm is
proportional to the number of living, metabolically active
cells.
10.sup.3 HCT116 cells were seeded per well. 24 hours later, the
medium was changed and the cells treated in triplicate with the
following concentrations of each compound: 10 .mu.M-3.33 .mu.M-1.11
.mu.M-370 nM-123 nM-41 nM-14 nM and 5 nM. The compounds were
diluted in 100% DMSO, whose final concentration on cells was kept
at 0.5%.
Cells were incubated with the compounds for 72 hours, and their
viability then assayed by the addition of MTS for 2 hours.
Absorbance at 492 nm was measured directly from the 96-well culture
plates. Gl50 (Growth Inhibition 50) concentrations for each
compound were calculated using a sigmoidal variable slope fit
(Prism 4.0, Graphpad Softwares). Values represent mean of 3
independent experiments.
PC3 Cell Viability and Proliferation Assay
PC-3 prostate cancer cells were obtained from ATCC, and maintained
in F-12K medium containing 7% FBS and 1% penicillin/streptomycin.
Cells were incubated at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2.
Cell viability was assayed using the MTS technique in 96-well
culture plates (CellTiter 96.RTM. Aqueous Non-Radioactive Cell
Proliferation Assay, Promega) according to the manufacturer's
instructions. MTS
(3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H-tetrazolium) is a MTT-derived tetrazolium that is reduced in
metabolically active cells into a soluble, cell-permeant formazan.
The amount of formazan, detected by its absorbance at 492 nm is
proportional to the number of living, metabolically active
cells.
2.times.10.sup.3 PC3 cells were seeded per well. 24 hours later,
the medium was changed and the cells treated in triplicate with the
following concentrations of each compound: 10 .mu.M-3.33 .mu.M-1.11
.mu.M-370 nM-123 nM-41 nM-14 nM and 5 nM. The compounds were
diluted in 100% DMSO, whose final concentration on cells was kept
at 0.5%.
Cells were incubated with the compounds for 72 hours, and their
viability then assayed by the addition of MTS for 2 hours.
Absorbance at 492 nm was measured directly from the 96-well culture
plates. Gl50 (Growth Inhibition 50) concentrations for each
compound were calculated using a sigmoidal variable slope fit
(Prism 4.0, Graphpad Softwares). Values represent mean of 3
independent experiments.
RESULTS
TABLE-US-00002 1. Inhibition of cysteine protease activities USP5
USP7 Experimental Experimental N.sup.o USP5 N.sup.o USP7 5a 1.8
.mu.M 5a 4 .mu.M 5b 1.15 .mu.M 5b 3.14 .mu.M 5d 1.42 .mu.M 5d 5.35
.mu.M 6 0.175 .mu.M 6 0.305 .mu.M 7 0.264 .mu.M 7 0.657 .mu.M 8a 54
.mu.M 8b 0.470 .mu.M 8b 0.226 .mu.M 8c 1.78 .mu.M 8c 0.470 .mu.M
10b 4.84 .mu.M 10f 1.2 .mu.M 10d 3.11 .mu.M 12 0.131 .mu.M 10f 3.25
.mu.M 13 0.215 .mu.M 10h 7.28 .mu.M 12 0.307 .mu.M 13 0.415 .mu.M
USP8 UCH-L3 Experimental Experimental N.sup.o USP8 N.sup.o Uch-L3
5a 0.58 .mu.M 5a 0.41 .mu.M 5b 0.355 .mu.M 5b 0.272 .mu.M 5c 47.7
.mu.M 5c 51 .mu.M 5d 0.565 .mu.M 5d 0.250 .mu.M 5e 35 .mu.M 5e 89
.mu.M 6 0.064 .mu.M 6 0.032 .mu.M 7 0.143 .mu.M 7 0.057 .mu.M 8a
27.8 .mu.M 8a 2.0 .mu.M 8b 0.121 .mu.M 8b 0.048 .mu.M 8c 0.225
.mu.M 8c 0.121 .mu.M 10b 0.528 .mu.M 10f 0.235 .mu.M 10d 0.381
.mu.M 12 0.044 .mu.M 10f 0.342 .mu.M 13 0.077 .mu.M 10h 0.807 .mu.M
12 0.037 .mu.M 13 0.071 .mu.M Cathepsine B Experimental N.sup.o
cathepB 5a 2.6 .mu.M 5d 6.7 .mu.M 6 0.300 .mu.M 7 0.890 .mu.M 8a
15.8 .mu.M 8b 2.1 .mu.M 8c 3.8 .mu.M 12 0.694 .mu.M 13 0.979 .mu.M
2. Inhibition of cell viability and proliferation HCT116 PC3
Experimental HCT116 Experimental PC3 N.sup.o GI50 D3 N.sup.o GI50
D3 5a 1.402 .mu.M 5a 6.15 .mu.M 5b 1.64 .mu.M 5b 6.69 .mu.M 5d 1.01
.mu.M 5d 2.79 .mu.M 6 0.096 .mu.M 6 0.180 .mu.M 7 0.363 .mu.M 7
0.466 .mu.M 8a 0.398 .mu.M 8a 0.391 .mu.M 8b 0.273 .mu.M 8b 0.457
.mu.M 8c 0.265 .mu.M 8c 0.502 .mu.M 10b 3.36 .mu.M 10f 8.4 .mu.M
10d 3.93 .mu.M 12 0.548 .mu.M 10f 2.1 .mu.M 13 1.97 .mu.M 10h 1.91
.mu.M 12 0.412 .mu.M 13 0.832 .mu.M
* * * * *